Purification of the Cucumber Necrosis Virus Replicase from Yeast Cells: Role of Coexpressed Viral RNA in Stimulation of Replicase Activity
Purification of the Cucumber Necrosis Virus Replicase from Yeast Cells: Role of Coexpressed Viral RNA in Stimulation of Replicase Activity
复制标题
从酵母细胞中纯化黄瓜坏死病毒复制酶：共表达病毒 RNA 在刺激复制酶活性中的作用
作者：
Z. Panaviene;T. Panavas;S. Serva;P. D. Nagy
摘要纯化的重组病毒复制酶可用于研究病毒RNA的体外复制机制。在本工作中，我们从酿酒酵母中获得了黄瓜坏死病毒(CNV)的高活性模板依赖复制酶复合体。CNV是一种正链RNA病毒。重组CNV复制酶表现出与植物来源的CNV复制酶(P.D.Nagy和J.Pogany，Viroology 276：279-288,2000)相似的性质，包括(I)在正链和负链模板上启动cRNA从头合成的能力，(Ii)通过内部起始产生比全长更短的复制酶产物的能力，以及(Iii)从模板的3‘端进行引物延伸的能力。我们还发现，功能复制酶的分离需要CNV p92RNA依赖的RNA聚合酶和辅助p33蛋白在酵母中共表达。此外，病毒RNA模板与复制酶蛋白在酵母中的共表达使纯化的CNV复制酶的活性增加了40倍，这表明病毒RNA可能促进了复制酶复合体的组装和/或RNA增加了复制酶的稳定性。综上所述，本文报道了第一个纯化的重组番茄病毒复制酶，该酶具有高活性和模板依赖性，这一发现将极大地促进未来RNA复制的体外研究。
ABSTRACT Purified recombinant viral replicases are useful for studying the mechanism of viral RNA replication in vitro. In this work, we obtained a highly active template-dependent replicase complex for Cucumber necrosis tombusvirus (CNV), which is a plus-stranded RNA virus, from Saccharomyces cerevisiae. The recombinant CNV replicase showed properties similar to those of the plant-derived CNV replicase (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), including the ability (i) to initiate cRNA synthesis de novo on both plus- and minus-stranded templates, (ii) to generate replicase products that are shorter than full length by internal initiation, and (iii) to perform primer extension from the 3′ end of the template. We also found that isolation of functional replicase required the coexpression of the CNV p92 RNA-dependent RNA polymerase and the auxiliary p33 protein in yeast. Moreover, coexpression of a viral RNA template with the replicase proteins in yeast increased the activity of the purified CNV replicase by 40-fold, suggesting that the viral RNA might promote the assembly of the replicase complex and/or that the RNA increases the stability of the replicase. In summary, this paper reports the first purified recombinant tombusvirus replicase showing high activity and template dependence, a finding that will greatly facilitate future studies on RNA replication in vitro.
天然和重组来源的脊髓灰质炎病毒 RNA 聚合酶的纯化、表征和比较。
DOI：
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发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Neufeld,KL;Richards,OC;Ehrenfeld,E
通讯作者：
Ehrenfeld,E